Artificial Nucleases for Targeted Single-Stranded DNA Breaks
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for genome editing, such as those using zinc finger nucleases (ZFNs), cannot induce targeted single-stranded breaks (SSBs) or facilitate their repair by homologous recombination in mammalian cells, limiting the ability to achieve targeted integration of transgenes without error-prone non-homologous end-joining (NHEJ) repair.
Innovation Solution
Development of artificial nucleases that generate targeted single-stranded breaks in double-stranded DNA using engineered zinc finger proteins with catalytically inactive cleavage half-domains, which facilitate homologous recombination and targeted integration by forming obligate heterodimers to create single-stranded nicks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If zinc finger nucleases are used to create double-strand breaks for genome editing, then targeted cleavage at specific genomic locations is achieved, but error-prone non-homologous end-joining repair occurs instead of homologous recombination
Solution Approach 1:
The invention divides the DNA cleavage function into two separate single-strand cleavage activities instead of one double-strand break. By using two different zinc finger nucleases that each cleave only one strand, the system creates a controlled single-strand break scenario that favors homologous recombination over error-prone NHEJ repair, thereby improving both reliability and precision of targeted integration
Solution Approach 2:
The invention changes the cleavage parameter from double-strand break to single-strand break by using catalytically inactive cleavage half-domains. This parameter change alters the cellular repair pathway preference from NHEJ to homologous recombination, enabling precise targeted integration while maintaining targeted cleavage capability
2Manufacturing precision
If catalytically inactive cleavage half-domains are used to form obligate heterodimers, then single-stranded breaks are generated for homologous recombination, but the device complexity increases
Solution Approach 1:
The invention merges two zinc finger nuclease components into an obligate heterodimer system where each component contains a catalytically inactive cleavage half-domain. This merging ensures that only when both components are present do they form a functional unit, reducing the need for separate delivery systems and simplifying the overall experimental procedure despite the increased molecular complexity
Solution Approach 2:
The catalytically inactive cleavage half-domains act as intermediaries that mediate the interaction between two zinc finger protein components. These intermediaries enable the formation of obligate heterodimers that generate single-strand breaks, facilitating homologous recombination while managing the complexity through controlled protein-protein interactions
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables precise and error-free targeted integration of transgenes into specific genomic locations in mammalian cells, avoiding the errors associated with non-homologous end-joining repair.
Implementation Method 1
artificial nucleases that generate targeted single-stranded breaks in double-stranded DNA using engineered zinc finger proteins with catalytically inactive cleavage half-domains
Data Source
AI summary
Disclosed herein are methods and compositions for generating a single-stranded break in a target sequence, which facilitates targeted integration of one or more exogenous sequences.